Pulmonary hypertension (PH) encompasses a diverse group of diseases marked by vascular injury and increased pulmonary artery pressures. Many sub-phenotypes of PH share a common link to the metabolic syndrome (systemic hypertension, obesity, glucose intolerance). For example, obesity, hyperlipidemia, and systemic hypertension are common risk factors for Group I pulmonary arterial hypertension (PAH) as well as for a subset of Group II (PH-heart failure with preserved ejection fraction, HF-pEF). The microbiome is important in development of obesity and the metabolic syndrome and is altered by high fat diet (HFD), suggesting that it may also play a role in PH. In Phase I of the tPPG, we tested 36 mouse strains and discovered that a 60% HFD led to PH HF-pEF, but only in certain strains. In our preliminary data, mice that developed PH on HFD had an increased ratio of gut Firmicutes to Bacteroidetes, similar to changes in obese humans with metabolic syndrome. In contrast, mice that did not develop PH on a high fat diet had a stable ratio. When commensal bacteria were decreased by treating mice with broad-spectrum antibiotics during HFD, the Firmicutes to Bacteroidetes ratio increased further, and manifestations of PH worsened. Despite a growing literature implicating the microbiome in a wide range of diseases, these studies are the first to support a critical protective role of bacteria in PH. Oral bacteria are also crucial, given that they are required for enterosalivary conversion of dietary nitrate to nitrite. Lack of these bacteria could result in decreased nitrite availability and resultant loss of downstream reactions in the host such as generation of nitric oxide and lipid signaling mediators such as electrophilic nitro-fatty acid (NO2-FA), leading to vasoconstriction and vascular proliferation. Abolishing oral microbes leads to systemic hypertension, but effects on the pulmonary vasculature are unknown. Thus, the goal of Project 3 is to define the relationships between PH, high fat diet, the oral and gut microbiome, and the metabolism of nitrogen oxide signaling mediators that may regulate the development and severity of PH. Our overall hypothesis is that the commensal microbiome is beneficial in PH because of its function in bioactivation of the nitrate-nitrite-NO-NO2-FA pathway. This hypothesis will be tested in murine models and in PH patients with the following specific aims: Aim 1. Bench To test the hypothesis that bacteria are essential for nitrate (NO3-) reduction and NOx metabolism in animal models of PH. Aim 2. Bedside To determine epidemiological relationships of oral and gut microbial structure and function to PH in humans and test the specific hypothesis that the oral microbiome is critical for protective nitrate bioactivation and improves pulmonary hemodynamics. Successful completion will profoundly shift the current paradigm of PH and yield novel avenues for therapy. This innovative proposal will leverage resources established during Phase I of the tPPG and is highly synergistic with all Projects and Cores.